Configurational stereoisomer of difethialone, composition and rodenticide bait comprising same, and method for controlling target rodent pests

ABSTRACT

Disclosed is a laevorotatory enantiomer of the configurational stereoisomer of difethialone, named homo-stereoisomer, the formula of which is 3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group have the same absolute configuration.

The invention relates to a configurational stereoisomer of difethialoneas an isolated compound, to a composition and a rodenticidal baitcomprising such a configurational stereoisomer and to a process forcontrolling target rodent pests. The invention thus relates to thetechnical field of controlling populations of target rodent pests.

It is known practice to use rodenticidal baits as poisons for targetrodent pests. It is known from EP 2 090 164 that difethialone is asecond-generation anticoagulant acting in a single dose. US 2005/181003describes a rodenticidal bait in gel form comprising difethialone in amass proportion of 25 ppm.

The baits of EP 2 090 164 and US 2005/181003 are liable to be consumedby animals other than target rodent pests when they are made availableto target rodent pests. They may be consumed directly (primaryconsumption) by domestic animals or pets. They may also be consumedaccidentally by humans. Such consumption may result in poisoning, whichmay be lethal, of these domestic animals or pets or of humans.

In addition, a fraction of the difethialone of these rodenticidal baitsmay be ingested (secondary consumption) by animals—especially bybirds—which prey on weakened rodent pests that have consumed such arodenticidal bait, or by animals which carrion-feed on rodent pests thathave died from having consumed such a rodenticidal bait. This secondaryconsumption is liable in the long term to result in the death of thesepredatory or carrion-feeding animals, which may be animals—especiallybirds—belonging to protected species.

The invention is thus directed towards overcoming these drawbacks byproposing a configurational stereoisomer of difethialone, a compositionand a rodenticidal bait comprising such a configurational stereoisomerand a process for controlling target rodent pests, which are not onlyeffective for controlling the populations of target rodent pests butalso limit the risks of poisoning of non-target animals—especiallydomestic or reared animals, pets or humans—which accidentally consumesuch a rodenticidal bait, and also the risks of poisoning, namedsecondary poisoning, of wild animals—for example foxes or birds—whichprey on weakened target rodent pests which have consumed therodenticidal bait or of wild animals which carrion-feed on target rodentpests that have died from being poisoned.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, the use of which complies with therules of environmental protection, especially with respect to theprotection of birds, and in particular birds of prey.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, which do not require, in order tocontrol a population of target rodent pests, the use of massive doses ofa rodenticidal agent and which are friendly towards the environment andthe health of humans and non-target animals—especially birds.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, which are able to be used forcontrolling target rodent pests that are resistant to known baits forcontrolling target rodent pests.

The invention is thus also directed towards proposing an alternative toknown rodenticidal baits.

To do this, the invention relates to a laevorotatory enantiomer of aconfigurational stereoisomer of difethialone, named homo-stereoisomer,the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group havethe same absolute configuration.

Throughout the text:

-   -   the term “difethialone” denotes the compound        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene        or        3-[3-[4-(4-bromophenyl)phenyl]-1-tetralinyl]-2-hydroxy-4-thiochromenone        or        3-[3-(4′-bromo[1,1′-biphenyl]-4-yl)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-hydroxy-2H-1-benzothiopyran-2-one        of formula (I) below:

in which are indicated the numbers of carbons 1 and 3 of the1,2,3,4-tetrahydronaphthalene group;

-   -   the term “stereoisomers” denotes isomers of the same        semi-structural formula, but in which the relative position of        the atoms differs in space. The term “configurational        stereoisomers” denotes stereoisomers for which conversion from        one to the other of these configurational stereoisomers requires        the cleavage/reformation of an interatomic covalent bond. Thus,        the term “configurational stereoisomers” denotes stereoisomers        which are not conformational isomers (or “rotamers”, for which        conversion from one to the other of the conformational isomers        is accompanied only by rotation of a part of the molecule about        the axis of a σ (sigma) bond formed by axial orbital overlap);    -   the term “homo-stereoisomer” of difethialone denotes the        configurational stereoisomer of difethialone of formula        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group have the same absolute configuration (1S,3S or 1R,3R),        said absolute configurations being determined according to the        sequential priority rules and the Cahn-Ingold-Prelog (CIP)        nomenclature;    -   the term “hetero-stereoisomer” of difethialone denotes the        configurational stereoisomer of difethialone of formula        3-(4′-bromobiphenyl-4-yl-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group have different absolute configurations (1S,3R or 1R,3S),        said absolute configurations being determined according to the        sequential priority rules and the Cahn-Ingold-Prelog (CIP)        nomenclature;    -   the term “amount” means a molar amount, a mass amount or a        volume amount. The proportions are thus proportions of a molar        amount relative to a molar amount, of a mass amount relative to        a mass amount, or of a volume amount relative to a volume        amount;    -   the term “substantially” indicates, in the usual manner, that a        structural or functional characteristic should not be taken as        marking an abrupt discontinuity, which would have no physical        meaning, but covers not only this structure or this function,        but also slight variations of this structure or of this function        which produce, in the technical context under consideration, an        effect of the same nature, or else of the same degree;    -   the expressions “high-pressure liquid chromatography” or        “high-performance liquid chromatography” (HPLC) denote “HPLC”        chromatography or “High Performance Liquid Chromatography”; and    -   the term “retention time” denotes the time, measured at the top        of the peak in the chromatogram, for which a compound is        retained on a chromatography column.

The invention thus relates to the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form. The inventionrelates to the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone in a form separated from the dextrorotatory enantiomer ofsaid homo-stereoisomer of difethialone and from the dextrorotatory andlaevorotatory enantiomers of a configurational stereoisomer ofdifethialone, named “hetero-stereoisomer”, in which carbons 1 and 3 ofthe 1,2,3,4-tetrahydronaphthalene group have different absoluteconfigurations.

The inventors have discovered that it is possible to separate thelaevorotatory and dextrorotatory enantiomers of said homo-stereoisomerof difethialone and of said hetero-stereoisomer of difethialone byhigh-pressure liquid chromatography in isocratic mode and underparticular conditions by using a chromatography column comprising achiral stationary phase. Specifically, it was not known at the date ofthe invention how to separate the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone and the dextrorotatory enantiomer ofsaid homo-stereoisomer of difethialone.

The inventors succeeded in performing this separation by choosing aparticular HPLC chromatography column, LUX® Cellulose-3 (Phenomenex, LePecq, France) of dimensions 150×2 mm and comprising a chiral stationaryphase constituted of porous particles of cellulosetris(4-methylbenzoate), having a particle size of 3 μm and a porosity of1000 Å. They used, as mobile phase, an eluent formed from a mixture ofacetonitrile (A) and water comprising formic acid in a volume proportionof 0.1% in water (B) with an A/B volume ratio of 80/20. The flow rate ofthe mobile phase in the column is kept at a value of 0.25 mL/minute andthe separation is performed at room temperature. The concentration ofthe composition to be analysed is 1 μg of difethialone per millilitre ofacetonitrile and the volume injected onto the column is 1 μL. Detectionmay be performed by tandem mass spectrometry (MS/MS). Detection may alsobe performed by photometry or by spectrophotometry by adjusting thedifethialone concentration and the injection volume for the purpose ofobtaining optimum detection and by measuring the value of the area underthe peak for each enantiomer.

Under these experimental conditions, the value of the retention time(t₁) for the laevorotatory enantiomer of said homo-stereoisomeraccording to the invention may vary depending on the operatingconditions—especially depending on the column temperature conditions—andmay be between 7.8 minutes and 8.2 minutes. The value of the retentiontime (t₄) for the dextrorotatory enantiomer of said homo-stereoisomermay vary depending on the operating conditions—especially depending onthe column temperature conditions—and may be between 14.0 minutes and14.4 minutes, such that the dextrorotatory and laevorotatory enantiomersof said homo-stereoisomer may be separated by high-pres sure liquidchromatography on a chiral column.

Under these same experimental conditions, the value of the retentiontime (t₃) for the dextrorotatory enantiomer of said hetero-stereoisomerof difethialone may vary depending on the operatingconditions—especially depending on the column temperature conditions—andmay be between 11.3 minutes and 11.8 minutes. The value of the retentiontime (t₂) for the laevorotatory enantiomer of said hetero-stereoisomerof difethialone may vary depending on the operatingconditions—especially depending on the column temperature conditions—andmay be between 9.0 minutes and 9.5 minutes.

Thus, under these experimental conditions, the order of elution of theconfigurational stereoisomers of difethialone is such that t₁<t₂<t₃<t₄.The retention time values t₁, t₂, t₃ and t₄ are liable to vary,especially with the temperature of the chromatography column. However,under these chromatographic conditions, the order of elution of theconfigurational stereoisomers of difethialone remains unchanged.

The invention thus relates to the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form and having theproperty of being able to be eluted, under the chromatography conditionsdescribed above, first from among the four configurational stereoisomersof difethialone.

The laevorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention, dissolved in methanolat a concentration of 0.81 g/L and placed in a quartz spectrophotometrycuvette, has a circular dichroism spectrum acquired at 25° C. withnegative circular dichroism values between 210 nm and 320 nm.

The laevorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention, dissolved inchloroform (CHCl₃), has a specific optical rotation [α]^(25° C.)_(589 nm), measured at 25° C. and on the sodium D line (589 nm), havinga value of −14.8°.

The laevorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention has, on proton magneticresonance (¹H-NMR) spectroscopy at 500 MHz in CDCl₃, a multiplet with achemical shift (δ) of between 4.9 ppm and 5.1 ppm corresponding to theproton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer of difethialone.

Said hetero-stereoisomer of difethialone and said homo-stereoisomer ofdifethialone are distinguished by their proton NMR spectra. In theproton NMR spectrum acquired in CDCl₃, the chemical shift of the protonborne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of saidhetero-stereoisomer of difethialone is between 5.2 ppm and 5.4 ppm,especially about 5.3 ppm.

The invention also relates to a composition comprising the laevorotatoryenantiomer according to the invention, with the exclusion of a racemicmixture of laevorotatory and dextrorotatory enantiomers of saidhomo-stereoisomer of difethialone, i.e. with the exclusion of acomposition in which the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone is in optically inactive mixture withthe dextrorotatory enantiomer of said homo-stereoisomer of difethialone.

The invention thus also relates to a composition comprising thelaevorotatory enantiomer of the configurational stereoisomer ofdifethialone, named homo-stereoisomer, of formula3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group havethe same absolute configuration, with the exclusion of a mixture inwhich the laevorotatory enantiomer and the dextrorotatory enantiomer ofsaid homo-stereoisomer of difethialone are in equal amounts. Theinvention thus relates to such a composition in which the laevorotatoryenantiomer and the dextrorotatory enantiomer of said homo-stereoisomerof difethialone are in different amounts.

Advantageously and according to the invention, said homo-stereoisomer ispredominantly in laevorotatory enantiomer form. Advantageously, acomposition according to the invention comprises said homo-stereoisomerof difethialone predominantly in laevorotatory enantiomer form.

Throughout the text, the term “said homo-stereoisomer is predominantlyin laevorotatory enantiomer form” means that the (mass, molar or volume)amount of laevorotatory enantiomer of said homo-stereoisomer ofdifethialone is in major amount—greater than 50%—in the total amount ofsaid homo-stereoisomer of difethialone present in the composition (inall its dextrorotatory and laevorotatory enantiomer forms).

Advantageously and according to the invention, the composition comprisesan amount of the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone such that the ratio of this amount to the amount of saidhomo-stereoisomer of difethialone in the composition is greater than50%, especially greater than 60%, in particular greater than 70%, moreparticularly greater than 80%, preferably greater than 90%, morepreferentially greater than 95%, particularly preferentially greaterthan 98%, even more preferentially greater than 99% or about 100%.Advantageously, the composition comprises an amount of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone such that the ratioof this amount to the amount of said homo-stereoisomer of difethialonein the composition is greater than 75%, preferably between 85% and 100%,more preferentially between 90% and 99%. Advantageously, the compositioncomprises an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone such that the ratio of this amount tothe amount of said homo-stereoisomer of difethialone is between 96% and100%.

In a composition according to the invention:

-   -   the amount of laevorotatory enantiomer of said homo-stereoisomer        of difethialone relative to the sum of the amounts of each of        the (laevorotatory and dextrorotatory) enantiomers of said        homo-stereoisomer of difethialone is greater than 0.5 (greater        than 50%);    -   the concentration of laevorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the sum of the        concentrations of each of the (laevorotatory and dextrorotatory)        enantiomers of said homo-stereoisomer of difethialone is greater        than 0.5 (greater than 50%); and    -   the proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone in the composition is greater        than the proportion of the other (dextrorotatory) enantiomer of        said homo-stereoisomer of difethialone. In a composition        according to the invention, the proportion of laevorotatory        enantiomer of said homo-stereoisomer of difethialone in the        composition is more than 50% relative to said homo-stereoisomer        of difethialone.

Advantageously, the composition may comprise a proportion ofdextrorotatory enantiomer of said homo-stereoisomer of difethialone,said proportion being less than 50%—especially less than 25%,preferentially between 0% and 25%, in particular less than 10%—relativeto the amount of said homo-stereoisomer of difethialone.

Advantageously and according to the invention, the difethialone ispredominantly in the laevorotatory enantiomer form of saidhomo-stereoisomer of difethialone. The composition comprises thelaevorotatory enantiomer of said homo-stereoisomer of difethialone in aproportion greater than each of the proportions of each of the otherenantiomers of difethialone relative to the difethialone.

Advantageously, in a composition according to the invention:

-   -   the ratio of the amount of laevorotatory enantiomer of said        homo-stereoisomer of difethialone to the sum of the amounts of        each of the enantiomers of said homo-stereoisomer of        difethialone and of said hetero-stereoisomer of difethialone is        greater than 0.25 (greater than 25%);    -   the ratio of the concentration of laevorotatory enantiomer of        said homo-stereoisomer of difethialone to the sum of the        concentrations of each of the enantiomers of said        homo-stereoisomer of difethialone and of said        hetero-stereoisomer of difethialone is greater than 0.25        (greater than 25%); and    -   the proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone in the composition is greater        than the proportion of each of the enantiomers of said        homo-stereoisomer of difethialone and of said        hetero-stereoisomer of difethialone. In a composition according        to the invention, the proportion of laevorotatory enantiomer of        said homo-stereoisomer of difethialone in the composition is        more than 25% relative to the difethialone.

Advantageously and according to the invention, the composition comprisesan amount of the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone such that the ratio of this amount to the amount ofdifethialone is greater than 25%, especially greater than 50%, inparticular greater than 70%, more particularly greater than 80%,preferably greater than 90%, particularly preferentially greater than95%, more preferentially greater than 98%, even more preferentiallygreater than 99% or about 100%. A composition according to the inventionthus comprises difethialone predominantly in the laevorotatoryenantiomer form of said homo-stereoisomer of difethialone.

Advantageously, the composition comprises an amount of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone such that the ratioof this amount to the amount of difethialone is greater than 70%,preferably between 80% and 100%, more preferentially between 90% and100%, relative to the amount of difethialone. Advantageously, thecomposition comprises an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone such that the ratio of this amount tothe amount of difethialone is between 95% and 99%. Advantageously, thecomposition comprises an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone such that the ratio of this amount tothe amount of difethialone is between 96% and 100%. Advantageously, thecomposition comprises an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone such that the ratio of this amount tothe amount of difethialone is substantially about 100%.

Advantageously and according to the invention, the composition comprisesan amount of the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone such that the ratio of this amount to the amount ofdifethialone is greater than 95%.

A composition according to the invention may be substantially free ofdextrorotatory enantiomer of said homo-stereoisomer of difethialone,i.e. the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone may be present in the composition, but only in traceamount. It may also be substantially free of said hetero-stereoisomer ofdifethialone, i.e. said hetero-stereoisomer of difethialone may bepresent in the composition, but only in trace amount.

Advantageously and according to the invention, the composition is inliquid form and comprises a liquid solvent for difethialone. It may be asolution of difethialone in a solvent for difethialone, with theexclusion of a racemic mixture of said laevorotatory and dextrorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolution comprising difethialone in a solvent for difethialone and inwhich said homo-stereoisomer of difethialone is predominantly inlaevorotatory enantiomer form. It may also be a solution comprisingdifethialone in a solvent for difethialone and in which the difethialoneis predominantly in the laevorotatory enantiomer form of saidhomo-stereoisomer of difethialone. It may also be a suspension or anemulsion of difethialone in a liquid medium.

Advantageously and according to the invention, the composition is insolid form. It may also be a solid comprising difethialone, with theexclusion of a racemic mixture of the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolid comprising difethialone and in which said homo-stereoisomer ofdifethialone is predominantly in laevorotatory enantiomer form. It mayalso be a solid comprising difethialone and in which the difethialone ispredominantly in the laevorotatory enantiomer form of saidhomo-stereoisomer of difethialone.

The invention thus also relates to a composition comprisingdifethialone, the difethialone of the composition being opticallyactive. However, it is not excluded for the difethialone of thecomposition according to the invention to be optically inactive.

The invention also relates to the use of a composition according to theinvention for the preparation of a rodenticidal bait for target rodentpests.

The invention also relates to a rodenticidal bait comprising acomposition according to the invention, and at least one excipient thatis edible for target rodent pests.

A rodenticidal bait according to the invention comprises:

-   -   at least one excipient that is edible for target rodent pests,    -   the laevorotatory enantiomer of the configurational stereoisomer        of difethialone, named homo-stereoisomer, of formula        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group of said homo-stereoisomer have the same absolute        configuration, with the exclusion of a racemic mixture of        laevorotatory and dextrorotatory enantiomers of said        homo-stereoisomer of difethialone. In a bait according to the        invention, the laevorotatory enantiomer of said        homo-stereoisomer of difethialone and the dextrorotatory        enantiomer of said homo-stereoisomer of difethialone are in        different amounts.

Advantageously, a rodenticidal bait according to the invention comprisesan excipient that is edible for target rodent pests and saidhomo-stereoisomer of difethialone predominantly in laevorotatoryenantiomer form.

The inventors who succeeded in separating the laevorotatory enantiomerand the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone and the enantiomers of said hetero-stereoisomer ofdifethialone and in obtaining the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form discovered that saidhomo-stereoisomer of difethialone is in fact the configurationalstereoisomer of difethialone that has the lower hepatic persistence intarget rodent pests. They also observed that the laevorotatoryenantiomer of said homo-stereoisomer of difethialone is theenantiomer—among the two enantiomers (laevorotatory and dextrorotatory)of said homo-stereoisomer of difethialone—that is the more persistent inthe liver of target rodent pests. However, they also discovered thatthis laevorotatory enantiomer of said homo-stereoisomer of difethialonehas higher persistence in the liver of target rodent pests than that ofsaid homo-stereoisomer of difethialone.

The laevorotatory enantiomer of said homo-stereoisomer of difethialonein fact makes it possible to efficiently control target rodent pests andin particular with reduced doses of difethialone. The laevorotatoryenantiomer of said homo-stereoisomer of difethialone constitutes analternative of choice for producing a rodenticidal bait which hasacceptable rodenticidal efficacy—especially at low dose—and acceptableecotoxicity. It also constitutes an alternative for producingrodenticidal baits for the purpose of controlling target rodent peststhat are liable to be resistant to known rodenticidal baits.

Advantageously and according to the invention, the rodenticidal baitcomprises a mass amount of difethialone such that the ratio (massproportion) of this mass amount of difethialone to the mass amount ofrodenticidal bait is less than 200 ppm, i.e. less than 200 mg ofdifethialone per kilogram of rodenticidal bait. Advantageously, the massproportion of difethialone in the rodenticidal bait is between 1 ppm and100 ppm (1 mg to 100 mg of difethialone per kilogram of rodenticidalbait), especially between 5 ppm and 100 ppm (5 mg to 100 mg ofdifethialone per kilogram of rodenticidal bait), preferably between 5ppm and 50 ppm (5 mg to 50 mg of difethialone per kilogram ofrodenticidal bait), more preferentially between 10 ppm and 50 ppm (10 mgto 50 mg of difethialone per kilogram of rodenticidal bait), even morepreferentially between 15 ppm and 50 ppm (15 mg to 50 mg of difethialoneper kilogram of rodenticidal bait).

Advantageously and according to the invention, the excipient that isedible for target rodent pests is chosen to allow consumption of thebait by target rodent pests. Advantageously and according to theinvention, each edible excipient is non-lethal to target rodent pests.The edible excipient is not in itself rodenticidal.

Advantageously and according to the invention, the edible excipientcomprises at least one food chosen from the group formed from cerealseeds—especially hulled cereal seeds—cereal seed meals, cereal seedflours, cereal seed flakes, cereal bran and non-cereal seeds, forexample alfalfa seeds, especially in hulled form, in the form of meal,in the form of flour, or in the form of flakes or bran. The edibleexcipient may comprise any support that can be consumed by target rodentpests.

Advantageously, the edible excipient comprises at least one food chosenfrom the group formed from foods of plant origin and foods of animalorigin. Advantageously, the edible excipient comprises at least one foodchosen to stimulate the appetite of the target rodent pests. Inparticular, this food is chosen from the group formed from seeds of oneor more cereals, hulled seeds of one or more cereals, meals of seeds ofone or more cereals, flakes of seeds of one or more cereals, bran of oneor more cereals and flour of seeds of one or more cereals. By way ofexample, the cereals are chosen from the group formed from oat, wheat,barley, corn, soybean and rice.

Advantageously, the food is chosen from the group formed from sweetenedfoods. For example, they may be foods comprising at least one sugarchosen from the group formed from sucrose, lactose, fructose andglucose. It may be a sugar syrup—for example a sugar syrup obtained byhydrolysis of starch—or a sugar syrup obtained by hydrolysis of sucrose(invert sugar syrup), or a beet sugar syrup, or a maple syrup or asugarcane syrup, or a syrup obtained from a plant of the Stevia genus.

Advantageously, the food is chosen from the group formed from coconutalbumen (copra) flakes and flour. Advantageously, the food is chosenfrom the group formed from walnuts, hazelnuts and almonds—in gratedand/or powder form. Advantageously, the food is chosen from the groupformed from plant fats, plant oils (for example rapeseed oil, soybeanfat, sunflower oil, cocoa butter, groundnut oil, groundnut butter, cornoil, palm oil), animal fats and animal oils (butter, lard, fish oil).

Advantageously, the food is chosen from the group formed from proteinsof plant origin and proteins of animal origin. By way of example,examples that may be mentioned include powdered milk—especially powderedskimmed milk—eggs—especially powdered eggs—protein hydrolysates ofanimal origin and protein hydrolysates of plant origin.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from solid baits comprising difethialoneand a solid edible excipient. Advantageously, the rodenticidal bait is asolid in divided form, for example in the form of balls or granules.Advantageously, the rodenticidal bait may be a solid in block or pasteform that may be consumed by the target rodent pests or a solid materialthat may be nibbled by the target rodent pests. Advantageously, thesolid rodenticidal bait according to the invention may be in the form ofa rigid block, a semi-rigid block, a foam, a powder or a gel.

Advantageously, the rodenticidal bait which is in the form of a powder,in the form of a foam or in the form of a gel is suitable for soilingthe fur of the target rodent pest(s) and for being ingested by saidpest(s) during their grooming.

It may be a solid rodenticidal bait comprising difethialone, with theexclusion of a racemic mixture of the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolid rodenticidal bait comprising difethialone and in which saidhomo-stereoisomer of difethialone is predominantly in laevorotatoryenantiomer form. It may also be a solid rodenticidal bait comprisingdifethialone in which the difethialone is predominantly in thelaevorotatory enantiomer form of said homo-stereoisomer of difethialone.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from liquid baits comprising difethialoneand a liquid edible excipient. The rodenticidal bait is then a drink fortarget rodent pests.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from liquid baits comprising difethialoneand a liquid edible excipient. The rodenticidal bait is then a drink fortarget rodent pests. It may be a solution of difethialone in a solventfor difethialone, with the exclusion of a racemic mixture ofdextrorotatory and laevorotatory enantiomers of said homo-stereoisomerof difethialone. It may also be a solution of difethialone in a solventfor difethialone and in which said homo-stereoisomer of difethialone ispredominantly in laevorotatory enantiomer form. It may also be asolution of difethialone in a solvent for difethialone and in which thedifethialone is predominantly in the laevorotatory enantiomer form ofsaid homo-stereoisomer of difethialone.

The invention thus also relates to a rodenticidal bait in which thedifethialone is optically active. However, it is not excluded for thedifethialone of the rodenticidal bait according to the invention to beoptically inactive.

Advantageously, the rodenticidal bait comprises at least one dye. Such adye makes it possible in particular to give said rodenticidal bait acolour that is readily detectable and identifiable by a person handlingthe rodenticidal bait.

Advantageously, the rodenticidal bait comprises at least one preservingagent capable of ensuring its conservation during its storage.Advantageously, the rodenticidal bait comprises at least one bitteringcompound such as denatonium benzoate, also known as Bitrex®, which isintended to reduce the risks of accidental consumption by non-targetorganisms.

Advantageously, in one particular variant, the composition and therodenticidal bait according to the invention exclusively comprisedifethialone as rodenticidal substance. In particular, the compositionand the rodenticidal bait according to the invention are free of anyother anticoagulant substance for rodenticidal use. However, in thisvariant according to the invention, the composition and the rodenticidalbait may comprise any pest-control substance other than a rodenticide,such as an insecticidal and/or acaricidal substance.

Advantageously, in another particular variant, the composition and therodenticidal bait according to the invention comprise difethialone withthe exclusion of a racemic mixture of laevorotatory and dextrorotatoryenantiomers of said homo-stereoisomer and at least one other substancedifferent from difethialone as rodenticidal substance. This otherrodenticidal substance different from difethialone may be anotheranticoagulant substance—especially of the anti-vitamin K type or not—oranother non-anticoagulant rodenticidal substance.

The invention also relates to a process for controlling target rodentpests, in which there is spread an amount of rodenticidal baitcomprising:

-   -   at least one excipient that is edible for target rodent pests;        and    -   the laevorotatory enantiomer of the configurational stereoisomer        of difethialone, named homo-stereoisomer, the formula of which        is        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group have the same absolute configuration;    -   with the exclusion of a racemic mixture of the laevorotatory and        dextrorotatory enantiomers of said homo-stereoisomer of        difethialone.

The invention also relates to a process for controlling target rodentpests, in which there is spread an amount of rodenticidal bait accordingto the invention, said amount of bait being sufficient to berodenticidal.

An amount of rodenticidal bait comprising said homo-stereoisomer ofdifethialone predominantly in laevorotatory enantiomer form, which hasacceptable rodenticidal efficacy even at a low dose of difethialone, isthus spread. The process according to the invention thus makes itpossible to limit the secondary poisoning of non-rodent mammals andbirds that are liable to feed on poisoned rodents which are dead oralive but comprising a reduced amount—and especially a non-lethalamount—of difethialone. The process according to the invention alsomakes it possible to limit such secondary poisoning of non-rodentmammals and birds that are liable to preferentially consume theviscera—in particular the liver—of said dead or live poisoned rodents.

Advantageously, in a process according to the invention, saidhomo-stereoisomer of difethialone is predominantly in laevorotatoryenantiomer form. Advantageously, the difethialone is predominantly inthe laevorotatory enantiomer form of said homo-stereoisomer ofdifethialone.

Advantageously and as a variant according to the invention, thefollowing are chosen in combination:

-   -   the edible excipient;    -   a proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone relative to said        homo-stereoisomer of difethialone;    -   a proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the difethialone;    -   a mass proportion of difethialone relative to the rodenticidal        bait; and    -   an amount of spread bait;    -   so that target rodent pests consume an amount of difethialone        that is sufficient to be lethal to said target rodent pests        which consume said bait in the course of a single period of 24        consecutive hours.

A rodenticidal bait according to this variant of the invention is a baitthat is mortal in a single intake, or a “one-shot” bait. Advantageouslyand according to this variant of the invention, the mass proportion ofdifethialone in the bait is between 5 ppm and 200 ppm, especiallybetween 5 ppm and 100 ppm, preferably between 5 ppm and 50 ppm, morepreferentially between 15 ppm and 50 ppm.

Advantageously and in another variant according to the invention, thefollowing are chosen in combination:

-   -   the edible excipient;    -   a proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone relative to said        homo-stereoisomer of difethialone;    -   a proportion of laevorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the difethialone;    -   a mass proportion of difethialone relative to the rodenticidal        bait; and    -   an amount of spread bait;        so that target rodent pests consume an amount of difethialone:    -   which is non-lethal to target rodent pests, i.e. which is        generally non-lethal to target rodent pests, which consume said        bait over a period of 24 consecutive hours; and    -   which is sufficient to be lethal to target rodent pests which        consume said bait over several 24-hour periods, said periods        being consecutive.

This other variant of the invention is thus directed towards a processfor controlling target rodent pests, in which there is spread an amountof rodenticidal bait that is lethal for target rodent pests whichdurably consume this rodenticidal bait and non-lethal for non-targetrodents or animals which accidentally consume this rodenticidal bait.This is then referred to as a “multi-dose” or “multi feeding” controlprocess. In such a process according to the invention, the consumptionof rodenticidal bait by a target rodent pest over a period of 24 hoursis insufficient to result in the death of said rodent, whereas repeatedconsumption of rodenticidal bait over at least two consecutive days canresult in the death of the target rodent pest. In addition, theaccidental consumption of rodenticidal bait by a non-target animal or ahuman over a period of 24 hours is usually insufficient to lead to thedeath of said animal or of a human.

The invention is thus directed towards a process for controlling targetrodent pests, in which target rodent pests are provided with an amountof rodenticidal bait that is liable to be ingested by the target rodentpests, said amount of rodenticidal bait being sufficient to kill targetrodent pests which consume said rodenticidal bait over several days.

Advantageously, the amount of rodenticidal bait spread, the massproportion of difethialone relative to the rodenticidal bait and theproportion of laevorotatory enantiomer of said homo-stereoisomer ofdifethialone relative to the difethialone are adapted so that theconsumption of the rodenticidal bait is lethal to target rodent pestswhich daily consume bait over at least two 24-hour periods, especiallyfrom 3 to 7 periods.

Advantageously, in this other variant of the invention, an amount ofrodenticidal bait is spread so that target rodent pests consume anamount of difethialone that is sufficient to be lethal to said targetrodent pests which consume said bait over several 24-hour periods, saidperiods being consecutive.

Advantageously, in this other variant of a process according to theinvention, the amount of laevorotatory enantiomer of saidhomo-stereoisomer of difethialone is such that since the ratio of thisamount to the amount of difethialone is greater than 95%, especiallyabout 100%, the mass proportion of difethialone relative to therodenticidal bait is between 5 ppm and 50 ppm, especially between 10 ppmand 15 ppm.

In a process according to the invention, target rodent pests areprovided with an amount of rodenticidal bait that is sufficient tosatisfy their daily appetite, said rodenticidal bait comprising a majorproportion of laevorotatory enantiomer of said homo-stereoisomer ofdifethialone.

In a process according to the invention, the amount of rodenticidal baitspread, the proportion of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone relative to the difethialone and themass proportion of difethialone relative to the rodenticidal bait areadapted so as to allow consumption of rodenticidal bait for several daysby target rodent pests, while at the same time limiting:

-   -   the risks of primary intoxication of non-target mammals and        birds which are liable to consume such a rodenticidal bait only        occasionally and accidentally;    -   the risks of secondary intoxication, for example of predators of        target rodents, which are liable to consume target rodents—dead        or live—that have ingested an amount of said bait.

Advantageously and according to the invention, the amount of baitspread, the ratio of the mass of difethialone to the mass of therodenticidal bait, the ratio of the amount of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone to the amount ofsaid homo-stereoisomer of difethialone and the ratio of the amount ofthe laevorotatory enantiomer of said homo-stereoisomer of difethialoneto the amount of difethialone are adapted to minimize the (total) amountof difethialone in the liver of the target rodent pests.

The invention also relates to a chromatographic process for obtaining alaevorotatory enantiomer of a configurational stereoisomer ofdifethialone, named homo-stereoisomer, the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration, in whichprocess:

-   -   a high-pressure liquid chromatography column of dimensions 150×2        mm, and comprising a chiral stationary phase constituted of        cellulose tris(4-methylbenzoate) particles, said particles        having a mean size of 3 μm and having a mean pore size of 1000        Å, is chosen;    -   a mixture formed from acetonitrile (A) and water comprising 0.1%        by volume of formic acid (B), with an A/B volume ratio of 80/20        and with a flow rate of the liquid mobile phase in the        chromatography column of 0.25 mL/minute, is chosen as liquid        mobile phase;    -   separation of the configurational stereoisomers of difethialone        is performed at room temperature, during which:    -   a liquid composition comprising said laevorotatory enantiomer of        said homo-stereoisomer of difethialone is introduced into the        top of the chromatography column; and then    -   the liquid composition is entrained with the mobile phase in the        chromatography column under conditions suitable for separating        the configurational stereoisomers of difethialone; and    -   a fraction of the mobile phase comprising said laevorotatory        enantiomer of said homo-stereoisomer of difethialone is        collected with a retention time t₁ having a value such that        t₁<t₂<t₃<t₄; t₂, t₃ and t₄ representing the retention times of        each of the configurational stereoisomers of difethialone        different from the laevorotatory enantiomer of said        homo-stereoisomer of difethialone, separately from a        dextrorotatory enantiomer of said homo-stereoisomer of        difethialone with a retention time t₄ and separately from the        laevorotatory and dextrorotatory enantiomers of a        configurational stereoisomer of difethialone, named        hetero-stereoisomer, in which carbons 1 and 3 of the        1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer        have different absolute configurations, and of retention times        t₂ and t₃; and then    -   the liquid mobile phase of said fraction is removed so as to        obtain said laevorotatory enantiomer of said homo-stereoisomer        of difethialone.

The invention also relates to a configurational stereoisomer ofdifethialone, to a process for obtaining such a configurationalstereoisomer, to a composition comprising such a configurationalstereoisomer, to a rodenticidal bait and to a process for controllingtarget rodent pests, which are characterized in combination by all orsome of the characteristics mentioned hereinabove or hereinbelow.

Other aims, characteristics and advantages of the invention will emergeon reading the following description and the examples, which are givenfor purely non-limiting purposes and which refer to the attachedfigures, in which:

FIG. 1 is a chromatogram of an analysis by high-pressure liquidchromatography on a chiral column of difethialone (top) and of thelaevorotatory enantiomer of said purified homo-stereoisomer ofdifethialone (bottom);

FIG. 2 is a proton NMR spectrum at 300 MHz of said homo-stereoisomer ofdifethialone;

FIG. 3 is a proton NMR spectrum at 300 MHz of said hetero-stereoisomerof difethialone;

FIG. 4 is a proton NMR spectrum at 500 MHz of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone;

FIG. 5 is a ¹³C carbon NMR spectrum at 500 MHz of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone;

FIG. 6 is an analysis by proton NMR (¹H-NMR) correlation spectroscopy at500 MHz of the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone;

FIG. 7 is a circular dichroism spectrum of the laevorotatory enantiomerof said homo-stereoisomer of difethialone;

FIG. 8 is a representation in graph form of the change over time of thehepatic concentration in rats (male and female) of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone (●) and of saidhomo-stereoisomer of difethialone (∘); and

FIG. 9 is a representation in graph form of the change over time of thehepatic concentration in rats (male and female) of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone (●) and of thetotal difethialone (Δ).

A. Purification of the Laevorotatory Enantiomer of said IsolatedHomo-Stereoisomer of Difethialone

A.1. Identification of said Homo-Stereoisomer of Difethialone

The homo-stereoisomer of difethialone is identified by proton magneticresonance (¹H-NMR) spectroscopy. The homo-stereoisomer of difethialonedissolved in CDCl₃ has a multiplet at a chemical shift (δ) of between4.9 ppm and 5.1 ppm and corresponding to the proton borne by carbon 1 ofthe 1,2,3,4-tetrahydronaphthalene group of difethialone as illustratedin FIG. 2.

The hetero-stereoisomer of difethialone and the homo-stereoisomer ofdifethialone are distinguished by their proton NMR spectra. In theproton NMR spectrum acquired in CDCl₃, the chemical shift of the protonborne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of saidhetero-stereoisomer of difethialone (FIG. 3) is between 5.2 ppm and 5.4ppm.

A.2. Separation of the Laevorotatory and Dextrorotatory Enantiomers ofsaid Homo-Stereoisomer of Difethialone by High-Pressure LiquidChromatography

The inventors solved the complex problem, which was not solved to date,of the separation of the laevorotatory and dextrorotatory enantiomers ofsaid homo-stereoisomer of difethialone from a commercial preparation ofdifethialone. They succeeded in performing an analytical separation ofthe enantiomers of said homo-stereoisomer of difethialone byhigh-pressure (high-performance) liquid chromatography on a LUX®Cellulose-3 chiral column

(Phenomenex, Le Pecq, France) of dimensions 150×2 mm and comprising achiral stationary phase constituted of porous particles of cellulosetris(4-methylbenzoate), with a particle size of 3 μm and a porosity of1000 A and using, as mobile phase, an eluent formed from a mixture ofacetonitrile (A) and water comprising formic acid in a volume proportionof 0.1% in water (B), with an A/B volume proportion of 80/20. The flowrate of the mobile phase in the column is 0.25 mL/minute and theseparation is performed at a temperature of 23.2° C. The solutioncontaining the sample to be analysed is at a concentration of 1 μg ofdifethialone per millilitre in acetonitrile and is filtered through aregenerated cellulose membrane with a cut-off threshold of 0.2 μm. Thevolume of solution containing the sample to be analysed injected ontothe column is 1 μL.

In a process for separating the enantiomers of said homo-stereoisomer ofdifethialone, it is possible to detect said enantiomers leaving thecolumn by tandem mass spectrometry (MS/MS) in negative electrosprayionization mode (ESI: ElectroSpray Ionization). The temperature of thecarrier gas is 350° C. and its flow rate is 8 L/minute. The pressure ofthe nebulizer is brought to 2700 hPa. In particular, the MRM (“MultipleReaction Monitoring”) transitions m/z 537.1→151.0 and m/z 537.1→78.9,corresponding to the difethialone signals, are analysed. FIG. 1represents the chromatograms of difethialone (top) and of thelaevorotatory enantiomer of the isolated homo-stereoisomer ofdifethialone (bottom).

Under these experimental conditions, the value of the retention time(t₁) for the laevorotatory enantiomer of said homo-stereoisomeraccording to the invention is about 8.1 minutes as described in FIG. 1.By way of comparison, the value of the retention time (t₄) for saiddextrorotatory enantiomer of said homo-stereoisomer according to theinvention is about 14.4 minutes, such that the dextrorotatory andlaevorotatory enantiomers of said homo-stereoisomer may be efficientlyseparated by high-pressure liquid chromatography on a chiral column.

The value of the retention time (t₃) for the dextrorotatory enantiomerof said hetero-stereoisomer of difethialone is about 11.7 minutes andthe value of the retention time (t₂) for the laevorotatory enantiomer ofsaid hetero-stereoisomer of difethialone is about 9.4 minutes. Thus,under these experimental conditions, the order of elution of thedifethialone enantiomers is such that t₁<t₂<t₃<t₄.

It is possible under these experimental conditions (stationary phase,mobile phase, temperature) to perform a preparative separation of thelaevorotatory and dextrorotatory enantiomers of said homo-stereoisomerof difethialone by using a similar stationary phase with a particle sizeof greater than 3 μm, and a chromatography column of larger dimensions,especially a diameter of 20 mm.

B. Structural Characterization

B.1. UV Spectroscopy

The UV spectrum of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in chloroform (CHCl₃) showsabsorbance peaks at 238.2 nm and 259.5 nm.

B.2. Optical Rotation

The inventors characterized the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form by means of itsoptical rotation (also known as the optical activity or circularbirefringence), i.e. its ability to deviate the polarization plane ofpolarized light. Deviation of the polarization plane of polarized lightclockwise facing the polarized light beam characterizes an opticallyactive and dextrorotatory solution and compound, and deviation of thepolarization plane of polarized light anticlockwise facing the polarizedlight beam characterizes an optically active and laevorotatory solutionand compound.

The optical rotation of a solution of laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in chloroform (CHCl₃) is measured at aconcentration of 11.05 g/L. The optical rotation of this solution ismeasured by means of a P 2000 digital polarimeter (JASCO, Bouguenais,France) operating with excitatory light with a wavelength of 589 nm. Themean optical rotation α obtained on two series of 10 measurements is−1.635° . The specific optical rotation at 25° C. _([α]) ^(25° C.)_(589 nm) for the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone dissolved in chloroform, measured on the sodium D line (589nm), is −14.8°.

B.3. Circular Dichroism

The circular dichroism spectrum of the laevorotatory enantiomer of saidisolated homo-stereoisomer of difethialone reflects the difference inabsorbance (ΔA=A_(L)-A_(R)) of the two waves of left circularpolarization (LCP) of intensity A_(L) and of right circular polarization(RCP) of intensity A_(R). This makes it possible to distinguish thedextrorotatory and laevorotatory enantiomers of said homo-stereoisomerof difethialone. This difference in absorbance of the two circularlypolarized waves is measured in a J-815 circular dichroism spectrometer(JASCO, Bouguenais, France). 2 mL of a solution of laevorotatoryenantiomer of said homo-stereoisomer of difethialone in methanol (CH₃OH)at a concentration of 0.81 mg/mL are prepared. The solution istransferred into a quartz spectrophotometer cuvette. The circulardichroism spectrum of the solution is measured at 25° C. between 200 nmand 400 nm. The circular dichroism spectrum of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone measured underthese conditions is shown in FIG. 7. The circular dichroism value isnegative between the wavelengths of 220 nm and 300 nm.

B.4. Nuclear Magnetic Resonance

FIGS. 2 and 3 represent, respectively, a proton nuclear magneticresonance spectrum (¹H-NMR) at 300 MHz of the homo-stereoisomer ofdifethialone in CDCl₃ (FIG. 2) and a proton nuclear magnetic resonancespectrum at 300 MHz in CDCl₃ of the hetero-stereoisomer of difethialonein CDCl₃ (FIG. 3). Said homo-stereoisomer of difethialone has (FIG. 2) amultiplet whose chemical shift (δ) is between 4.9 ppm and 5.1 ppm,corresponding to carbon 1 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer of difethialone. Said hetero-stereoisomer ofdifethialone has (FIG. 3) a multiplet whose chemical shift (δ) isbetween 5.2 ppm and 5.4 ppm.

FIG. 4 is a proton NMR spectrum at 500 MHz of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone in CDCl₃. Theproton

NMR spectra of the dextrorotatory and laevorotatory enantiomers of saidhomo-stereoisomer of difethialone are indistinguishable from each other.

FIG. 5 is a ¹³C NMR spectrum of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in CDCl₃ at a concentrationof 40 mg/mL, acquired on a Bruker Avance III HD spectrometer (500 MHz)equipped with a Prodigy motorized multi-core direct cryoprobe. It allowsidentification of the 31 carbon atoms of difethialone. The ¹³C-NMRspectrum of the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone is not distinguished from the ¹³C-NMR spectrum of thedextrorotatory enantiomer of said homo-stereoisomer of difethialone.Said homo-stereoisomer of difethialone has a characteristic signalbetween 34 ppm and 38 ppm which is distinctive for saidhetero-stereoisomer of difethialone.

FIG. 6 is a two-dimensional proton NMR (2D ¹H-NMR) spectrum obtained bycorrelation spectroscopy of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in CDCl₃ at a concentrationof 40 mg/mL acquired on a Bruker Avance III HD spectrometer (500 MHz)equipped with a Prodigy motorized multi-core direct cryoprobe. It allowsidentification of the coupling of the proton borne by carbon 1 of the1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer ofdifethialone with the protons borne by carbon 2 of the1,2,3,4-tetrahydronaphthalene group at 2.27 ppm.

C. Extraction of Difethialone from the Liver of Rats Treated withDifethialone for the Purpose of Analysis of the Various Enantiomers ofDifethialone

C.1. Homogenization of the Liver Sample

About 0.525 g (±0.025 g) of rat liver is weighed out accurately andplaced in a 50 mL polypropylene tube. 10 mL of acetone are added and thesuspension is homogenized using an Ultra-Turrax® homogenizer/disperserfor a time of about 30 seconds. The homogenizer/disperser shaft isrinsed with hot water and then twice with 20 mL of acetone in apolypropylene tube. The homogenate is centrifuged for 5 minutes at acentrifugation speed of 3000 rpm (revolutions per minute). Thesupernatant is collected and transferred into a test tube. The sample issubjected to evaporation under a stream of nitrogen (N₂) at atemperature of 40° C. so as to form a dry extract.

C.2. Lipid Removal

1 mL of acetonitrile is added to the tube containing the dry extract soas to dissolve it. The acetonitrile solution is washed twicesuccessively with 1 mL of hexane. The lipid-free extract is dried undera stream of nitrogen (N₂) at a temperature of 40° C. and is then takenup in 0.5 mL of methanol and dissolved by vortex stirring. 0.5 mL ofultra-pure (Milli-Q) water is then added. The sample isvortex-homogenized.

C.3. Solid-Phase Extraction (SPE) of Difethialone

1 mL of dichloromethane (CH₂Cl₂), then 1 mL of methanol (CH₃OH), then 1mL of ultra-pure (Milli-Q) water are passed through an Oasis HLB 1 cccartridge (WAT094225, Waters). The lipid-free liver extract (1 mLMeOH/Milli-Q H₂O) containing difethialone is then loaded onto the top ofthe preconditioned cartridge. The liver extract penetrates through thecartridge by gravity on contact with the solid phase of the cartridge. 1mL of washing solution formed from methanol (CH₃OH) and ultra-pure water(H₂O) in a 90/10 volume proportion is loaded onto the top of thecartridge. The cartridge is dried by suction under vacuum connected tothe bottom of the cartridge. 1 mL of eluting solution formed fromdichloromethane (CH₂Cl₂) and methanol (CH₃OH) in a 90/10 volumeproportion is then loaded onto the top of the cartridge and an eluatecomprising difethialone is collected at the bottom of the cartridge. Thesolvent of the eluate is evaporated off under a stream of nitrogen (N₂)at a temperature of 40° C. The sample is taken up in 0.5 mL ofacetonitrile (NC-CH₃) and the acetonitrile solution containingdifethialone is filtered through a 0.2 μm filter.

C.4. Analysis

The acetonitrile solution containing difethialone is analysed byhigh-pressure liquid chromatography on a LUX® Cellulose-3 chiral column(150×2 mm, particle size of 3 μm) (Phenomenex, Le Pecq, France) asdescribed in point A2) above. The retention time value (t₁) of thelaevorotatory enantiomer of said homo-stereoisomer of difethialoneaccording to the invention is between 7.8 minutes and 8.4 minutes (themaximum value of the peak corresponding to the dextrorotatory enantiomerof said homo-stereoisomer being about 8.1 minutes, as described in FIG.1), depending on the operating conditions, especially depending on thecolumn temperature conditions. The retention time value (t₄) of thedextrorotatory enantiomer of said homo-stereoisomer is between 13.3minutes and 15.1 minutes (the maximum value of the peak corresponding tothe dextrorotatory enantiomer of said homo-stereoisomer being about 14.4minutes), depending on the operating conditions, especially depending onthe column temperature conditions. The retention time value (t₃) of thedextrorotatory enantiomer of said hetero-stereoisomer of difethialone isbetween 11.2 minutes and 12.3 minutes (the maximum value of the peakcorresponding to the dextrorotatory enantiomer of saidhetero-stereoisomer being about 11.7 minutes), depending on theoperating conditions, especially depending on the column temperatureconditions. The retention time value (t₂) of the laevorotatoryenantiomer of said hetero-stereoisomer of difethialone is between 9.0minutes and 9.8 minutes (the maximum value of the peak corresponding tothe laevorotatory enantiomer of said hetero-stereoisomer being about 9.4minutes), depending on the operating conditions, especially depending onthe column temperature conditions. The composition of the sample isanalysed by high-pressure liquid chromatography as described in pointA2) above.

D. Study of the Hepatic Persistence of the Configurational Stereoisomersof Difethialone in Rats

A solution of a mixture of said homo-stereoisomer and of saidhetero-stereoisomer of difethialone in a mixture of vegetable oil and 5%DMSO is administered by tube-feeding (“per os”) to male and femalecoumaphen-sensitive rats (Rattus norvegicus). The mole ratio of theamount of said homo-stereoisomer to the amount of saidhetero-stereoisomer is 4/6. Each difethialone diastereoisomer is formedfrom a racemic mixture of the two enantiomers of the correspondingdiastereoisomer.

The tube-feeding solution is administered (on DO) so that the amount ofdifethialone ingested by each rat is about 3.4 mg per kilogram of rat.

To avoid haemorrhage, the tube-fed rats are treated daily bysubcutaneous administration of a dose of vitamin K1 (as haemorrhageantidote) at a rate of 0.1 U per 200 g of live rat weight.

At 4 hours (H+4), 9 hours (H+9), 24 hours (H+24), 120 hours (H+120), 168hours (H+168) and 216 hours (H+216) after tube-feeding, three male ratsand three female rats anaesthetized beforehand with isoflurane areeuthanized, the liver of the euthanized rats is removed, thedifethialone is then extracted from the liver and the amount of each ofthe configurational stereoisomers of difethialone is assayed viaanalysis by high-pressure liquid chromatography on a chiral columnaccording to the process described above, the area under the peaks inthe chromatogram obtained is measured and each enantiomer is quantifiedby comparison with a calibration curve. The following are assayed:

-   -   the dextrorotatory enantiomer of said homo-stereoisomer;    -   the laevorotatory enantiomer of said homo-stereoisomer according        to the invention;    -   the dextrorotatory enantiomer of said hetero-stereoisomer;    -   the laevorotatory enantiomer of said hetero-stereoisomer;        present in the liver of the tube-fed rats.

In FIGS. 8 and 9, the difethialone concentrations recorded (mean of thevalues measured on six rats and expressed in nanograms of enantiomer pergram of liver (ng/g) in the liver of the tube-fed rats) are given as afunction of the time (in hours) after tube-feeding. In FIG. 8, theconcentration of the laevorotatory enantiomer of said homo-stereoisomeraccording to the invention in the liver is represented by filled circles(●) on the left-hand scale and the concentration of saidhomo-stereoisomer of difethialone in the liver is represented by opencircles (∘) on the right-hand scale.

The laevorotatory enantiomer of said homo-stereoisomer according to theinvention has inexplicably high hepatic persistence relative to thepersistence of the homo-stereoisomer of difethialone.

In FIG. 9, the concentration of the laevorotatory enantiomer of saidhomo-stereoisomer according to the invention in the liver is representedby filled circles (●) on the left-hand scale and the total difethialoneconcentration in the liver is represented by open triangles (Δ) on theright-hand scale. The corresponding values are given in table 1 below.

TABLE 1 Hepatic content, ng/g Time Total difethialone afterHomo-stereoisomer Hetero-stereoisomer tube-feeding, DFN-Homo- DFN-Homo-DFN-Hetero- DFN-Hetero- hours dextro laevo dextro laevo 4 4566 5692.510589.5 5380.5 9 4692.5 7141 12155.5 5869.5 24 1243.5 4403.5 8102 3613.548 720.5 3874 7974.5 2804 120 192.5 2087 5431.5 1211 168 129 878.5 3011392.5 216 77.5 1224 4030.5 545

The laevorotatory enantiomer of said homo-stereoisomer according to theinvention has high hepatic persistence relative to the persistence ofdifethialone.

Rodenticidal Bait Comprising a Proportion of 13.7 ppm of Difethialone

A pasty rodenticidal bait according to the invention is prepared bydispersing an amount of laevorotatory enantiomer of saidhomo-stereoisomer of difethialone in an edible excipient comprisingvegetable fat and cereal flour. The measured proportion of difethialonerelative to the bait is 13.7 ppm and the proportion of laevorotatoryenantiomer of said homo-stereoisomer relative to the difethialone is95.4% (13.1 mg of laevorotatory enantiomer of said homo-stereoisomer ofdifethialone per kilogram of bait). The bait also comprises a massproportion of 3.3% of dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone relative to the difethialone and amass proportion of 1.4% of dextrorotatory enantiomer of saidhetero-stereoisomer of difethialone relative to the difethialone.

On D0, ten coumaphen-sensitive Sprague-Dawley (SD) rats (five male ratsand five female rats) are placed in individual cages with arodenticide-free reference feed. On D3, each rat is weighed, and 50 g ofrodenticidal bait as described above are then provided to each rat. Thisprovision of 50 g of rodenticidal bait is renewed daily. The baitconsumed by the rats is made up to 50 g of bait on D4, D5 and D6. Theweight gain of each of the rats is also measured daily. Starting fromD7, the residual rodenticidal baits are removed and rodenticide-freefeed is provided to all the rats. The rats are monitored for 3 weeks.

The mean amounts of bait consumed daily by a rat on D4, D5, D6 and D7expressed in grams per day are given in table 2 below.

TABLE 2 Bait consumed Mean Standard deviation D4 17.8 5.7 D5 16.0 4.7 D614.2 4.1 D7 9.4 3.8

It should be noted that no rat consumed a daily amount of bait of lessthan 1 g/day. All the rats (100%) die between D9 and D10. The mortalityis 100% on D10.

The bait containing a low dose of 13.7 ppm of difethialone makes itpossible to obtain a mortality rate of 100% while at the same timeminimizing the risks of intoxication of animals—especially birds—whichprey or carrion-feed on weakened target rodent pests that have consumeda rodenticidal bait according to the invention.

It goes without saying that the invention may be the subject of numerousimplementation variants and applications. In particular, a composition,a rodenticidal bait and a process for controlling target rodent pestsare subject to an infinite number of variants both in the formulation ofthe bait and in the embodiments of the process.

1. Laevorotatory enantiomer of a configurational stereoisomer ofdifethialone, named homo-stereoisomer, the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group havethe same absolute configuration.
 2. Composition comprising thelaevorotatory enantiomer according to claim 1, with the exclusion of aracemic mixture of laevorotatory and dextrorotatory enantiomers of saidhomo-stereoisomer of difethialone.
 3. Composition according to claim 2,wherein said homo-stereoisomer is predominantly in laevorotatoryenantiomer form.
 4. Composition according to claim 2, wherein thedifethialone is predominantly in the laevorotatory enantiomer form ofsaid homo-stereoisomer of difethialone.
 5. Composition according toclaim 2, wherein an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone is such that the ratio of this amountto the amount of difethialone is greater than 25%.
 6. Compositionaccording to claim 2, wherein an amount of the laevorotatory enantiomerof said homo-stereoisomer of difethialone is such that the ratio of thisamount to the amount of difethialone is greater than 95%. 7.Rodenticidal bait comprising a composition according to claim 2 and atleast one excipient that is edible for target rodent pests.
 8. Baitaccording to claim 7, wherein the edible excipient comprises at leastone food chosen from the group formed from cereal seeds, cereal seedmeals, cereal seed flours, cereal seed flakes, cereal bran andnon-cereal seeds.
 9. Bait according to claim 7, wherein a mass amount ofdifethialone is such that the ratio of this mass amount of difethialoneto the mass amount of rodenticidal bait is less than 200 ppm. 10.Process for controlling target rodent pests, in which there is spread anamount of rodenticidal bait comprising: at least one excipient that isedible for target rodent pests; and a laevorotatory enantiomer of theconfigurational stereoisomer of difethialone, named homo-stereoisomer,the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group havethe same absolute configuration; with the exclusion of a racemic mixtureof the laevorotatory and dextrorotatory enantiomers of saidhomo-stereoisomer of difethialone.
 11. Process according to claim 10,wherein said homo-stereoisomer of difethialone is predominantly inlaevorotatory enantiomer form.
 12. Process according to claim 10,wherein the difethialone is predominantly in the laevorotatoryenantiomer form of said homo-stereoisomer of difethialone.
 13. Processaccording to claim 10, wherein the following are chosen in combination:the edible excipient; a proportion of laevorotatory enantiomer of saidhomo-stereoisomer of difethialone relative to said homo-stereoisomer ofdifethialone; a proportion of laevorotatory enantiomer of saidhomo-stereoisomer of difethialone relative to the difethialone; a massproportion of difethialone relative to the rodenticidal bait; and anamount of spread bait; so that target rodent pests consume an amount ofdifethialone that is sufficient to be lethal to said target rodent pestswhich consume said bait in the course of a single period of 24consecutive hours.
 14. Process according to claim 10, wherein thefollowing are chosen in combination: the edible excipient; a proportionof laevorotatory enantiomer of said homo-stereoisomer of difethialonerelative to said homo-stereoisomer of difethialone; a proportion oflaevorotatory enantiomer of said homo-stereoisomer of difethialonerelative to the difethialone; a mass proportion of difethialone relativeto the rodenticidal bait; and an amount of spread bait; so that targetrodent pests consume an amount of difethialone: which is non-lethal totarget rodent pests which consume said bait over a period of 24consecutive hours; and which is sufficient to be lethal to target rodentpests which consume said bait over several 24-hour periods, said periodsbeing consecutive.
 15. Chromatographic process for obtaining alaevorotatory enantiomer of a configurational stereoisomer ofdifethialone, named homo-stereoisomer, the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration, in whichprocess: a high-pressure liquid chromatography column of dimensions150×2 mm, and comprising a chiral stationary phase constituted ofcellulose tris(4-methylbenzoate) particles, said particles having a meansize of 3 pm and having a mean pore size of 1000 Å, is chosen; a mixtureformed from acetonitrile (A) and water comprising 0.1% by volume offormic acid (B), with an A/B volume ratio of 80/20 and with a flow rateof the liquid mobile phase in the chromatography column of 0.25mL/minute, is chosen as liquid mobile phase; separation of theconfigurational stereoisomers of difethialone is performed at roomtemperature, during which: a liquid composition comprising saidlaevorotatory enantiomer of said homo-stereoisomer of difethialone isintroduced into the top of the chromatography column; and then theliquid composition is entrained with the mobile phase in thechromatography column under conditions suitable for separating theconfigurational stereoisomers of difethialone; and a fraction of themobile phase comprising said laevorotatory enantiomer of saidhomo-stereoisomer of difethialone is collected with a retention time t₁having a value such that t₁<t₂<t₃<t₄; t₂, t₃ and t₄ representing theretention times of each of the configurational stereoisomers ofdifethialone different from the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone, separately from a dextrorotatoryenantiomer of said homo-stereoisomer of difethialone with a retentiontime t₄ and separately from the laevorotatory and dextrorotatoryenantiomers of a configurational stereoisomer of difethialone, namedhetero-stereoisomer, in which carbons 1 and 3 of the1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer havedifferent absolute configurations, and of retention times t₂ and t₃; andthen the liquid mobile phase of said fraction is removed so as to obtainsaid laevorotatory enantiomer of said homo-stereoisomer of difethialone.16. Composition according to claim 3, wherein the difethialone ispredominantly in the laevorotatory enantiomer form of saidhomo-stereoisomer of difethialone.
 17. Composition according to claim 3,wherein an amount of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone is such that the ratio of this amountto the amount of difethialone is greater than 25%.
 18. Compositionaccording to claim 4, wherein an amount of the laevorotatory enantiomerof said homo-stereoisomer of difethialone is such that the ratio of thisamount to the amount of difethialone is greater than 25%. 19.Composition according to claim 3, wherein an amount of the laevorotatoryenantiomer of said homo-stereoisomer of difethialone is such that theratio of this amount to the amount of difethialone is greater than 95%.20. Composition according to claim 4, wherein an amount of thelaevorotatory enantiomer of said homo-stereoisomer of difethialone issuch that the ratio of this amount to the amount of difethialone isgreater than 95%.